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Plant Breeding for Disease Resistance in Modern Agriculture

The world today, have a very great demand for plant breeding. This is a very magnificent technology that is used to change the trait character of the plant species. Changing the trait in the characteristic of the plant plays a major role in today’s modern agriculture sector. Plant breeding techniques also help contribute to the incredible efficiency of modern agriculture. This contribution is made on many aspects such as in soil, climates but the most important is in the disease resistance. In plant breeding, the breeder utilizes their knowledge in making the best feature of two different parents’ plant while selecting them in the best possible way to combine them. Different books indicate that Mendel is known as the father of genetics. Till date, it is very important for plant breeding especially in the Australian continent. Initially when the breeding program had been introduced in Australia, the main aim of breeders was to maintain a higher yield and to make the crop drought resistant; whereas now, this now have been moved to breed the disease resistant variety in different cultivating aspects. Plant breeding in 1890 was established by John Garton; yet it successfully came under commercialization in the late nineteenth century. Plant breeding is a broad-spectrum that has many steps involved in the breeding. Collection of the germplasm, evaluation and selection of the parent, cross hybridization in selected plants, selection of characters in the parents, emasculation bagging and tagging are some of the steps involved in plant breeding. However, in the modern agriculture genetic modification technology (GM) is used. It is a technology that helps in altering of genes (inserting or deleting genes) between the same or different species but in an in-vitro (inside laboratory) conditions. Apart from diseases, in agriculture is insect and the pests. They feed on the parts of the plants like leaves, stems or flowers making the plant very easily susceptible for diseases. In general, insect infestation on the plants leads to yield loss and also the quality of the product. In-order to protect the plants from pest farmers use pesticides. This not only kills the harmful pest but also the useful ones. There are some crop varieties that’s breeders developed through hybridization and selection for insect pest resistance. There are different steps involved in plant breeding. They are; collection of different plants from different regions, evaluation and selection of parents, hybridization or mixing, selection of superior recombinant by growing the seeds initially and testing and releasing of new cultivars obtaining the desired seed with the required trait. Plant breeding not just play a vital role in the crop yield but also in the disease resistance. Crops in-order to give a good and a very high potential yield needs to be disease free or disease resistance. This includes the fungus, bacteria and the virus which are pathogens. The capacity of the plant to block pathogens are the host resistant plant. This is possible through plant breeding. With the process of plant breeding, it results in the reduction of the cost input for the production making it cost reasonable for not just a farmer but also to the consumers. Disease resistant plants are above all very safe for human beings as it does not contain any artificial synthesis or pesticides that is applied on the plant. Additionally, plant breeding reduces hazards or environmental pollution and on living being hazard which are impacted by the use of harmful pesticides on the plant crop. This is why plant breeding has a very high advantage in the disease resistance. The modern agriculture is under a very high pressure. The population is growing very high. The food consumption is really getting onto the sly limit. Each and individual farmers need feeding more and more people daily but with the same amount of land where the production is limiting every year despite a very hard effort. The amount of food produced in last centuries is the same that we will be needing to produce in the next years to come. This is in inside earth climate change, drought and pests. More and more farmers are migrating leaving more barren lands. In future we tend in having less lands with which we ought to make more food. We can only achieve it with more seeds. Plant breeding has been making plants stronger with providing greater yield with pest resistance, early maturity and with a better quality making them easier to harvest. Research has been the key but yet more of the research is needed. Plant breeders breed better seeds which is yet somehow bringing a negative long-term result by reducing the footprint of agriculture. There are many types of trait locus and molecular markers that are available. However, those that are chosen depends on many factors. Among them, co-dominant is highly preferable. Polymorphism is necessary for genetic mapping not the physical mapping. Currently, molecular markers where there is sequence for the information are preferred to anonymous markers. For the effective of sharing PCR is used. It is very common for the plant breeders to come up with necrosis during their breeding process which has a potential to abandon sensitive varieties from breeding. Despite these facts, using effectors has a very wide range of benefits. Some of them are; a quick rapid assay, cost effective, saves a lot of places, operating cost are kept minimum, breeders tend in knowing the exact effector causing symptom on the plant. A condition which is not normal and has an alternating effect that brings an abnormal growth in a plant which is termed as a plant disease. In the past, there were many cases which were termed as epidemic which brought the total loss of the plant crop in terms of yield and production which were either by natural calamities such as drought, famine and so on. On the other hand, farmers are often hurdled by the disease pest in the plant crop and this has often bought huge concerns and debates among farmers and the researchers or the plant breeder. Therefore, researchers or plant breeders are actively involved in making a plant disease resistant. They have created some varieties that are drought resistant and disease resistant as well. This has brought plant breeding and breeders to a new era.
Plant breeding has often been encountered as a very difficult and a lengthy process despite the steps in the breeding show a very simpler step. Earlier, plant breeding was done in the traditional method which consumed a lot of time; mostly it took years to breed at least a variety. This was mainly done for the varietal development. But today, with the advancement of the technology, this can be done in a very quicker time, Additionally, this breeding technology also had capacities in changing the genetic composition of the plants. But today, plant breeding is not just about conducting experiments or breeding on the field for varieties rather it is for the disease resistance of plant crops. Often crops are attacked by harmful pest which cause a very high impact on the resistance of the plant which is used to remove the unwanted traits or character of the plant and insert a new useful trait into the plant system. This is all done inside the laboratory condition. With the highest level of technology present today, such as PCR, we can easily operate the equipment without any interferences.
Case Study:
It is very important and vital that plant breeding take place from time to time for both farmers and the breeders (company) to survive. Most of the breeding’s are done in collaboration between the breeding company and the farmers while some are solely done in the laboratory conditions. Plant breeding basically not just maintains the diversity in keeping it in a good condition but also has a higher capacity in making the plant disease resistant.
Another ample example of plant breeding is the one done in the molecular level. This has a very highly equipped machine which changes the DNA sequence of a plant by either inserting or deletion of a gene in the plant crop. For instance, in wheat plant, the loss of yield from fungal disease is mainly cause my P. nodurum and P. tritici-repentis which brings about a loss of approximately $ 1.7 billion in the Australian economy. There are cases that represents that’s plant breeding has highly been very advance in the recent years which the innovation of various technology such as DNA fingerprinting that is very fast and cost effective. Above all, this technology requires no pre-information for the process of investigation.

Also, in the wheat plant it has been observed that disease trait was removed from the crop and inserted a resistant gene. The most cost effective and a convenient way for the breeders for effector sensitivity is the gene resistant. This can either be done by marker assisted breeding or by effector assisted breeding. Effector sensitivity for gene resistant in the wheat plant can me mapped towards the chromosome of the wheat for the disease resistance. Example, SnTox3-Snn3 infiltrates the effector proteins in the wheat plant that is very sensitive to the necrosis or the death of a cell in the susceptible wheat variety. This has brought many varieties of the wheat crop in commercialization in the western Australia. Also, from the haploid population of the wheat plant double haploid population from e intergrain were released. Therefore, breeding for different plant disease in the crop using the molecular marker especially in the wheat crop have had a very handy and a positive result in disease resistance of the wheat plant. Breeding for a better resistance plant for disease resistance in plant crops in regard to the plant crops.
Plant breeding has become one of the most popular method for breeding the hybrid crops in different environmental condition. Drought resistance, lack of water or nitrogen stress are very important for the agriculture. Plant breeding has been in practice since the beginning of the agriculture. In every country plant breeding is necessary for the sustainable agriculture practice that produces stress free crops helping in the rise of the economy of the country. Plant breeding is very useful for making the crop more resistant with plant breeding, alteration of the genes could bring a disease-free plant or help in removing the harmful disease character traits from the plant and inserting the useful genes to make the plant crop more resistant to factors such as disease, drought and son and so for. Also, when there is a high number of resistant genes in a plant crop especially supporting the disease resistance the market value automatically gets risen. In other words, there is a very high market value for the resistant crop variety. Plant breeding has an advantage if increasing the yield of a crop plant. This can be only possible when there is a disease-free crop resulting in aftermath of a high yield. Plant breeding is all about domestication involving such as seed dormancy, improve seed yield and a disease resistant crop and improved food adaption for its uses. Domesticated crop that are grown by breeders cannot survive on their own and needs an artificial support. Breeders breed the plant such as corn in the green revolution are some examples that farmers use and apply rapidly. It is often said that the genetic makeup of the plants could be modified to a much greater extent than we normally appreciate. however, breeding of several crop plants, like pulses and vegetables, has not yet been so intensive as that of wheat and rice. There is much more modification that are to be done on these crops on the basis of their yield. The worlds result has also sown from the past that there are many losses in the yield of the plant crop with several factors that intend to reduce the yield of the crop. For example, climate, disease, soil and so on and so for. For these in the coming year, many detailed researches are to be carried out and investigate them with a proper finding and a sustainable solution. Also, additionally, in-order to achieve these future prospects, crop biotechnology could also accelerate the plant breeding in disease resistance in the modern agriculture practices.
Reference:
Jain, K. (2018). Plant Breeding: Steps and Methods of Plant Breeding for Disease Resistance! [online] Biology Discussion. Available at: http://www.biologydiscussion.com/plants/plant-breeding-steps-and-methods-of-plant-breeding-for-disease-resistance/1340.
Yang, H., Tao, Y., Zheng, Z., Li, C., Sweetingham, M. and Howieson, J. (2012). Application of next-generation sequencing for rapid marker development in molecular plant breeding: a case study on anthracnose disease resistance in Lupinus angustifolius L. BMC Genomics, [online] 13(1), p.318. Available at: http://www.biomedcentral.com/1471-2164/13/318.
Faris, J., Zhang, Z., Lu, H., Lu, S., Reddy, L., Cloutier, S., Fellers, J., Meinhardt, S., Rasmussen, J., Xu, S., Oliver, R., Simons, K. and Friesen, T. (2010). A unique wheat disease resistance-like gene governs effector-triggered susceptibility to necrotrophic pathogens. Proceedings of the National Academy of Sciences, 107(30), pp.13544-13549.
Tanksley, S. (1983). Molecular Markers in Plant Breeding. Plant Molecular Biology Reporter, 1: 1 (1983), pp.3-8.
Moose, S. and Mumm, R. (2008). Molecular Plant Breeding as the Foundation for 21st Century Crop Improvement. PLANT PHYSIOLOGY, 147(3), pp.969-977.
McDonald, B. and Linde, C. (2002). PATHOGENPOPULATIONGENETICS, EVOLUTIONARYPOTENTIAL, ANDDURABLERESISTANCE. Annual Review of Phytopathology, 40(1), pp.349-379.
Vanloqueren, G. and Baret, P. (2008). Why are ecological, low-input, multi-resistant wheat cultivars slow to develop commercially? A Belgian agricultural ‘lock-in’ case study. Ecological Economics, 66(2-3), pp.436-446.
Smithson, J. and Lenne, J. (1996). Varietal mixtures: a viable strategy for sustainable productivity in subsistence agriculture. Annals of Applied Biology, 128(1), pp.127-158.

A Three Point Test Cross in Drosophilia: Recombination and Linkage

Abstract
A three-point testcross was performed with Drosophila to distinguish the location and relationship of three genes (y cv f) on the X chromosome. The parental is a virgin female triply mutant and a wild-type male. This testcross includes mating a trihybrid wild-type F1 female to a mutant F1 male. One hundred F2 offspring were then scored and 8 different phenotypes were observed and assigned reciprocal classes. The order of the three genes was then determined to be (y cv f). Recombination frequencies for crossovers in both region 1 and region 2 were calculated for three data sets. A Chi Square examination utilizing the real data (published recombination frequencies) was then directed to decide the exactness of the test recombination frequencies for crossovers in region 1, 2, and the reciprocal crosses for all the data set collections. A linkage map was drawn from the recombination frequencies for region 1 and 2. A linkage map recombination rates to delineate the physical distance between genes on a chromosome. The linkage map related reasonably with the recombination frequencies, anyway there were inconsistencies with the frequency of single crossovers.
Introduction
Gene mapping is very significant in genetics because of several reasons. Location of the gene provides information about its function, structure, and location. Gene mapping can determine the gene is transmitted from maternal or paternal side. It can also determine how many genes caused an illness transmitted from a parent to kid by using recombination percentage. Gene mapping in particular gives the location of the gene and it is important to make complex DNA sequences and genomes.
During the Prophase 1 the crossing over takes place between the tetrads. The probability of crossing over increases in the genes that are farther apart. Linkage happens when at least two or more genes are found close-by one another on a same chromosome. In this situation, the genes don’t assort independently and they inherited together, as a result, the ratio is different than Mendelian Ratios. Recombination frequency (Rf) is the rate for the number of recombinants out of the total number of progeny in one region. A gene map evaluates the physical distance between two gene loci utilizing information from the measure of recombination. Recombination Frequency can’t larger than 50 % and linked genes are less than 50%.
It is beneficial to map three genes at once rather than mapping each gene separately. Mapping three genes at once, gives information about the order and the distance between the genes. This enables understudies to distinguish genes on a gene map despite the fact that it isn’t sure whether the sequence is perused from appropriate to left or the other way around. Mapping each pair of gene doesn’t give accurate information about the distance and the order of the genes.
A standout amongst the most imperative life forms that have been utilized to ponder hereditary qualities for quite a few years is Drosophila melanogaster, additionally called the “Fruit Fly,” known to immediately emerge within the sight of matured organic product. They are exceptionally helpful for hereditary investigation since they breed effectively, they have short life expectancies and generation periods, and they’re anything but difficult to keep up and control with respect to sustenance and temperature. Likewise, natural product flies are little enough to gather extensive populaces yet sufficiently expansive to recognize wild compose attributes from mutant characteristics.
The Hypothesis for this experiment is RF measured in lab will be similar to the expected RF based on known map distances. Reciprocal classes will occur and survive in equal numbers. Interference will be a positive value.
Methods:
The Experiment started with the cross of two fly: wild type male ( ) and completely mutant virgin females (wfm). The pale coloring, the folded wings, the enlarged abdomens can use to distinguish virgin females, and they have a meconium. Using virgin females in all crosses is very important. Female drosophila can just mate with one male, and they at that point store that male’s sperm for the rest of their adult life. In this manner, with a specific end goal to control mating, females must be separated not long after they eclose from their pupa casing, while despite everything they show the highlights recorded above, and have an ensured virgin status. The parental flies were removed and the F1 generation is ready to be crossed. The second cross involves mating 4 heterozygous wild type females to 6 F 1 mutant males. After one week students will score the F2 generation for each of the three traits. This cross is performed to decide if the genes assort independently. If the genes assort independently the frequency for all the phenotypes would be 12.5% in the F generation. If the parental phenotypes are shown most of the time, or far more prominent than the normal 12.5% (i.e. no requirement for chi squared examination), at that point it can be assumed that genes are linked. Since linked genes are on a same chromosome, it shows that the genes are inherited together most of the time, and it clarifies the absence of independent assortment. Also, the other phenotypes are the result of crossing overs during meiosis. The following phenotypes and Genotypes were observed for F2 progeny: Grey color, cross vein, straight bristles ( ), yellow color, crossveinless, forked bristles (ycvf), Grey, crossveinless, forked bristles ( cvf), yellow , crossvein, straight bristles (y ), Grey, crossvein, and forked bristles( f), yellow color, crossveinless, straight bristles (ycv ),Grey color, crossveinless, straight bristles( cv ), and yellow color, crossvein, forked bristles.(y f). The parental phenotypes which are the non-recombinants are the most numerous in a three-point test cross. Double crossover classes in F2 are usually the least frequent classes of progeny. To conclude which gene is the middle of the three genes, a comparison between non-recombinants and double crossover classes is made. Non-recombinants have parental genotypes , and ycvf and double cross over classes has the following genotypes cv , y f. By comparing these two classes you can distinguish the order of gene in middle because in double cross overs the middle gene gets inverted from its initial position in the parental chromosome. Single crossover classes in region 1 had recombinant genotypes differing at the first gene : cvf, y . Single cross over classes in region two had recombinant genotypes differing at the third gene : f, ycv .
Three different traits were recorded for the F2 Drosophila. Y= yellow color body. The wings exhibit a yellow glint in this mutant due to lack of dark melanin pigments. The wild flies are grey color and express melanin pigments.
Cv=crossveinless. The mutant is lacking crossveins in the wing, whereas the wild fly has prominent veins. f=forked bristles. The mutants have shorter bristles with bent segments on the head and thorax regions. Wild flies have longer, straight bristles on their head and thorax regions.
It is unnecessary to record the sex of the F2 since within each of the 8 F2 progeny classes, both genders are phenotypically the same. The F1 males are all mutants because they cannot undergo crossing over because in males the X chromosome can’t make a homolog with the Y chromosome. On the other hand, these three genes are located on the X-chromosome not Y so F1 males are hemizygous.
Recombination Frequency is calculated by the division of the sum of all classes of progeny resulting from crossover in one region by the total number of progeny. One percent recombination is the equivalent to 1 map unit. A linkage map uses the recombination frequencies to determine distance between two gene locations. Each crossover has effect on another crossover and they interfere with each other.
C.O.C is calculated by the sum of the observed double cross overs divided by expected number of double crossovers. An interference of value 1 means there wasn’t any crossovers and the interference was complete. The value of 0 means the observed number of crossovers is equal to the number of expected crossovers. The value of interference between 0 and 1 means some interference happened.
There are 18 different Chi Square calculated for 6 data sets.
1.Chi Square for crossovers in region 1: expected vs observed
2.Chi Square for crossovers in region 2: expected vs observed
3.Chi Square for parental reciprocal cross similarity: expected vs observed
4.Chi Square for single crossovers in region 1 reciprocal cross similarity: expected vs observed
5.Chi Square for single crossovers in region 2 reciprocal cross similarity: expected vs observed
6.Double crossovers reciprocal cross similarity: expected vs observed.
Results:
Actual y _______13.7 ______cv _____43_______ f
Small data y _______16.07 ______cv _____17.85_______ f
Class data y _______16.4 ______cv _____32.83_______ f
Expert data y _______15.4 ______cv _____30_______ f
Four linkage maps are shown. The three data sets have different Rf values between the three genes (y cv f ) when compared to published data. The published RF for distance between y-cv is =13.7cM. The published Rf for the distance between genes cv-f is =43cM.
Classes
Genotypes
Expert Data
Small data
Class data
Non-recombinant (Parental)

420
35
249
Non-recombinant (Parental)
ycvf
326
4
134
Single cross over in Region 1
y
70
3
28
Single cross over in Region 1
cvf
63
4
35
Single cross over in Region 2
ycv
162
3
83
Single cross over in Region 2
f
161
5
89
Double Cross Overs
y f
33
0
23
Double Cross Overs
cv
29
2
23
total
1264
56
664
Interference(I)
0.0446551
0.245053
0.28669
Calculation:
RF= (70 63 33 95)/(1264)=0.154 * 100= 15.4%
C.O.C= (33 29)/(1264*0.1542*0.3045)=1.0446551
Interference=1-c.o.c=1-1.0446551=0.0446551
cross overs in region one : expected vs observed
0.2944562
P>0.05
0.266365
P>0.05
4.141793
P<0.05
cross overs in region two: expected vs observed
80.73505
P<0.05
16.56805
P<0.05
28.0126
P<0.05
parental reciprocal cross similarity: expected vs observed
11.84
P<0.05
24.346
P<0.05
34
P<0.05
single cross over in region 1 reciprocal cross similarity: expected vs observed
0.368
P>0.05
0.1428
P>0.05
0.77
P>0.05
single cross over in region 2 reciprocal cross similarity: expected vs observed
0.00315
P>0.05
0.5
P>0.05
0.209
P>0.05
Double cross reciprocal cross similarity: expected vs observed
0.258
P>0.05
2
P>0.05
0
P>0.05
This table includes the Calculated 18 Chi Square tests for the three-point testcross experiment. Interference and c.o.c are also calculated for each data set.
Discussion:
Usually, traits on non-homologous chromosomes are inherited independently of each
other’s locations. However, traits that are linked on homologous chromosomes are often inherited together unless crossing over between the homologous chromosomes occurs. (Klug et. al. 2012) The hypothesis for this experiment was RF measured in lab will be similar to the expected RF based on known map distances. Reciprocal classes will occur and survive in equal numbers. Interference will be a positive value. Based on the results from the experiment the hypothesis is wrong and rejected.
For the parental reciprocal cross there is a similarity in all three data sets and the p0.05. For the single crossovers in region 2, the data shows the similar X2 and the p value is less than 0.05. For the double cross overs the X2 is different but still they have p value greater than 0.05. For the class data set X2=4.14 but for small data and the expert data the X2 is similar and the p value is greater than 0.05. For single cross overs in region 2 reciprocal cross similarity the class data set and small data set are similar but the expert data set is different. Reciprocal classes from single cross overs in region 2 and double cross overs are selected against and do not survive in in equal numbers. Based on the prediction the reciprocal class non-recombinants had the most frequencies, while the double cross overs occurred the least. Single cross overs in region 1 and region 2 occur in similar numbers for small data and expert data but for class data the frequency is less and it is almost equal to double cross overs.
The data shows the frequency for crossovers in region one is less than region two and it is because the Rf for region one is less than the Rf in region 2. The measurements for Rf for region one is similar in all three data sets but the calculated Rf value in region is vary for all three data. For the expert data set, all but the reciprocal parental cross for reciprocal classes occurred and survived in equal numbers. For the expert data set, interference had a positive value. For the small data set, recombination frequency for crossovers in region 1 measured in lab was similar to the expert and class data but they are different than actual Rf value. For the small data set Interference is a positive value.
For the class data set the Rf measured in lab was not similar to the expected Rf but it is similar to small data and expert data.. This occurrence does not occur by chance, it is likely due to miscoding of the flies. Missing double crossovers that appear to be similar to non-recombinants may have also caused the error.
It is difficult to correctly measure long map distances by recombination frequency because of recombination. If two genes have a small recombination frequency, there will be less chance for recombination to occur. For more accurate measurements of long map distances two genes must be located farther apart from each other on the same chromosome. When two genes located further away from each other, there is higher chance for recombination to occur.
There are some errors could happened during the experiment. First by anesthetized the flies two times, some of the flies could have died. By putting the flies in the jar, some of them might wedge in the food and died. This mistake reduced the sample size and made it difficult to score phenotypes of some of the flies covered with food. The higher the number of the flies causes the percentage error to decrease. RF measured in lab will be similar to the expected RF based on known
map distances. The Rf value was not similar for all the three data sets. The first part of hypothesis is rejected. Reciprocal classes will occur and survive in equal numbers, is true for all three data sets. The interference is positive for all three data sets.
Conclusion:
The data shows that a three-point testcross can be used to locate the positions of three linked genes on the X chromosome. The Chi square analysis is significant to show the occurrence of recombination in different regions. Experimental data such as recombination frequencies can then be used to map out the physical distance between these genes on the same chromosome. Statistical analysis of Chi-square and map gene can determine the ratio of the progeny, the percent of the crossovers.
Reference:
Aggarwal, D. D., Rashkovetsky, E., Michalak, P., Cohen, I., Ronin, Y., Zhou, D., … Korol, A. B. (2015). Experimental evolution of recombination and crossover interference in Drosophila caused by directional selection for stress-related traits. BMC Biology, 13, 101. doi: 10.1186/s12915-015-0206-5

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